effects of cymbopogon sp. essential oils and streptomycin sulfate on ...

EFFECTS OF CYMBOPOGON SP. ESSENTIAL OILS AND

STREPTOMYCIN SULFATE ON ANTIMICROBIAL PROTEINS

PRODUCTION BY BACILLUS SUBTILIS ATCC 21332

Hairul Shahril Bin Muhamad

UNIVERSITI SAINS ISLAM MALAYSIA

EFFECTS OF CYMBOPOGON SP. ESSENTIAL OILS AND

STREPTOMYCIN SULFATE ON ANTIMICROBIAL PROTEINS

PRODUCTION BY BACILLUS SUBTILIS ATCC 21332


(Matric No.: 3100018)

Thesis submitted in fulfillment for the degree of

MASTER OF SCIENCE

Faculty of Science and Technology

UNIVERSITI SAINS ISLAM MALAYSIA

NILAI

MARCH 2014

i

AUTHOR DECLARATION

بسم هللا الرحمن الرحيم

I hereby declare that the work in this thesis is my own except for quotations and

summaries which have been duly acknowledged.

Date: 31 March 2014 Signature :

Name : Hairul Shahril Bin Muhamad

Matric No : 3100018

Address : No. 19, Kg. Sikota, Air Mawang,

73100 Johol, N. Sembilan.

ii

APPROVAL

This thesis entitled “Effects of Cymbopogon sp. Essential Oils and Streptomycin

Sulfate on Antimicrobial Proteins Production by Bacillus subtilis ATCC 21332”

submitted to the Faculty of Science and Technology, USIM and was accepted as

fulfillment of the requirements for the degree of Master of Science.

………………...................

HANINA MOHD NOOR, Ph.D,

Faculty of Science and Technology,

Universiti Sains Islam Malaysia

Date: 31 March 2014

iii

DEDICATION

To

My wife and my child for the motivation and love:

Nurul Atiqah & Muhammad Adam Haris

My parents and my parents in law for the kindness and support:

Muhamad & Fatimah

and

Nizam & Rosmidah

My brothers and sisters for the inspiration

iv

BIODATA OF AUTHOR

Hairul Shahril Bin Muhamad (3100018) was born on the 27th

January 1986 at Hospital

Besar Tampin, Tampin N. Sembilan. Currently, he is residing at Nilai, N. Sembilan.

He performed his primary education at Sekolah Kebangsaan Nuri, Johol, N. Sembilan

and furthered his secondary education at SMKA Sheikh Haji Mohd. Said, Seremban,

N. Sembilan. He continued his study in matriculation level at Malacca Matriculation

College. On June 2005, he joined Islamic Science University of Malaysia (USIM) as

an undergraduate student in the Faculty of Science and Technology and graduated in

August 2009 with Bachelor of Science with Honoured (Food Biotechnology). Now he

attached with Islamic Science University of Malaysia (USIM) again as a graduate

student majoring in Master of Science (Food & Biotechnology).

v

ACKNOWLEDGEMENTS

In the name of Allah, the most Compassionate and the most Merciful. Praise is to

Allah, Lord of the universe for giving me the strength to endure all challenges and

complete this study. Also peace and prayers be upon His Prophet and Messenger.

My sincere appreciation goes out to Prof. Dr. Bachok bin M. Taib, Dean of the

Faculty of Science and Technology and my supervisor, Dr. Hanina Binti Mohd. Noor

for their great concern and persistence encouragement. I am heartily thankful to my

supervisor for her supervision, advice, and guidance from the very early stage of this

research as well as giving me extraordinary experiences throughout the work. She has

providing answers and ideas to my never-ending stream of questions. Above all and

the most needed, she provided me unflinching encouragement and support in various

ways. Her truly scientist intuition has made her as a constant oasis of ideas and

passions in science, which exceptionally inspire and enrich my growth as a student, a

researcher and a scientist want to be. I am indebted to her more than she knows.

Love and thanks to my beloved lovely parents, wife, brothers and sisters for their love

and constant supports. I thank them for simply being there and loving me with all their

hearts. Special thanks also goes out to laboratory assistance of Microbiology Lab at

Faculty of Science and Technology (Mr. Fredy, Mr. Mazlan and Miss Sarina) for their

assistance and guidance and other members of the faculty, whose help and

cooperation were invaluable during the course of the study. Collective and individual

acknowledgments are also owed to my colleagues at Microbiology Lab whose present

somehow perpetually refreshed, helpful, and memorable.

Finally, I would like to thank everybody who was important to the successful

realization of this thesis, as well as expressing my apology that I could not mention

personally one by one.

vi

ABSTRAK

Kesan Minyak Pati Cymbopogon sp. dan Streptomisin Sulfat terhadap

Penghasilan Protein Antimikrob oleh Bacillus subtilis ATCC 21332


March 2014

Penghasilan protein oleh bakteria mungkin meningkat dalam persekitaran yang

tertekan, contohnya dengan kehadiran agen antimikrob. Didapati kebanyakan agen

antimikrob, apabila digunakan pada kepekatan yang rendah, menunjukkan keupayaan

untuk mengaktifkan atau merencatkan transkripsi gen, yang mana ianya berbeza

daripada kesan perencatan oleh agen tersebut. Walau bagaimanapun, terdapat hanya

beberapa kajian mengenai potensi sebatian semula jadi di dalam alam ini selaku

isyarat kimia spesifik yang boleh mencetuskan pelbagai fungsi biologi. Oleh itu,

tujuan kajian ini adalah untuk menilai kesan minyak pati Cymbopogon sp. (iaitu C.

nardus dan C. flexuosus) dan Streptomisin Sulfat dalam mengawal penghasilan

protein atau peptida oleh Bacillus subtilis ATCC 21332. Keputusan micropencairan

menunjukkan bahawa Kepekatan Perencatan Minimum (MICs) C. nardus dan C.

flexuosus serta Streptomisin Sulfat ke atas B. subtilis ATCC 21332 adalah 1.56%

(v/v), 0.2% (v/v) dan 2.5 mg/ml masing-masing. Minyak pati C. flexuosus dan C.

nardus pada kepekatan 0.01 MIC, setiapnya telah ditambah semasa fasa awal log

pertumbuhan bakteria pada suhu 37ºC, menyebabkan pengeluaran protein intrasel

baru dengan saiz yang sama dalam lingkungan saiz 180 kDa yang mana dikenalpasti

sebagai enzim ‘DNA-directed RNA polymerase’ subunit β dan enzim ‘respiratory

nitrate reductase’ subunit α masing - masing. Selain itu, apabila B. subtilis ATCC

21332 telah didorong oleh minyak pati C. flexuosus dan Streptomisin Sulfat semasa

fasa log tumbesaran sel pada suhu 30ºC juga boleh merembeskan protein ekstrasel

dengan saiz anggaran 30 kDa, dikenal pasti sebagai Bacillopeptidase F. Tambahan

pula, B. subtilis ATCC 21332 dengan kehadiran sama ada minyak pati C. nardus atau

C. flexuosus boleh merembeskan protein ekstrasel bioaktif dengan keupayaan aktiviti

antimikrob terhadap bakteria Gram-positif dan Gram-negatif tertentu. Oleh itu, B.

subtilis ATCC 21332 dalam keadaan tertekan dengan kehadiran sama ada minyak pati

Cymbopogon sp. atau Streptomisin Sulfat pada aras 0.01 MIC dapat mendorong

pengeluaran atau perembesan protein bioaktif.

vii

ABSTRACT

Effects of Cymbopogon sp. Essential Oils and Streptomycin Sulfate on

Antimicrobial Proteins Production by Bacillus subtilis ATCC 21332


March 2014

Proteins level produced by bacteria may be increased in stressful surroundings, such

as in the presence of antimicrobial agent. It appears that many antimicrobial agents,

when used at low concentrations, have in common the ability to activate or repress

gene transcription, which is distinct from their inhibitory effect. However, there have

been comparatively few studies on the potential of natural compounds as a specific

chemical signal that can trigger a variety of biological functions. Therefore, the aim of

this study is to evaluate the effects of Cymbopogon sp. (which are C. nardus and C.

flexuosus) essential oils and Streptomycin Sulfate in regulating proteins or peptides

production by Bacillus subtilis ATCC 21332. Results of Microdilution assay showed

that the Minimum Inhibition Concentrations (MICs) of C. nardus and C. flexuosus as

well as Streptomycin Sulfate on B. subtilis ATCC 21332 were 1.56% (v/v), 0.2% (v/v)

and 2.5 mg/ml respectively. C. flexuosus and C. nardus essential oils at concentration

of 0.01 MIC, each was added during early log phase of bacterial growth at 37ºC,

resulting the production of new intracellular proteins with similar approximate size of

180 kDa in which recognized as DNA-directed RNA polymerase β subunit enzyme

and respiratory nitrate reductase α subunit enzyme respectively. Besides, when B.

subtilis ATCC 21332 were induced by C. flexuosus essential oil and Streptomycin

Sulfate during log phase of growing cells at 30ºC could also secrete the extracellular

proteins with approximate size of 30 kDa, identified as Bacillopeptidase F. In

addition, B. subtilis ATCC 21332 in the presence of either C. nardus or C. flexuosus

essential oils could secrete the bioactive extracellular proteins with potent

antimicrobial activity against certain Gram-positive and Gram-negative bacteria.

Hence, B. subtilis ATCC 21332 in stressful condition with the presence of either

Cymbopogon sp. essential oils or Streptomycin Sulfate at 0.01 MIC level were able to

induce the production or secretion of bioactive proteins.

viii

ملخص البحث

على Streptomycin Sulfate الزيوت األساسية و ليرة sp. Cymbopogonآثار

Bacillus subtilis ATCC 21332الببتيدات بواسطة البروتينات مضادات الميكروبات

خيرالشهريل بن محمد

4102 مارس

ويمكن زيادة مستوى البروتينات التي تنتجها البكتيريا في محيط مرهقة، كما هو الحال في وجود

ويبدو أن العديد من العوامل المضادة للجراثيم، عند استخدامها . عامل مضاد للميكروبات

بتركيزات منخفضة، وتشترك في القدرة على تنشيط أو قمع النسخ الجيني، والذي يختلف من

ومع ذلك، فقد كانت هناك دراسات قليلة نسبيا على إمكانات المركبات الطبيعية . المثبطةتأثيرها

. في الطبيعة كإشارة كيميائية معينة التي يمكن أن تؤدي مجموعة متنوعة من الوظائف البيولوجية

والتي هي . )ليرة سورية Cymbopogonولذلك، فإن الهدف من هذه الدراسة هو تقييم آثار

C.nardus وC. flexuosus ) الزيوت األساسية وكبريتات الستربتوميسين في تنظيم إنتاج

وقد تحددت تركيزات تثبيط الحد . 23112سي سي البروتينات بواسطة العصوية الرقيقة آي تي

الزيوت األساسية وكذلك C. flexuosusو C. nardusمن ( البلدان المتوسطة الدخل)األدنى

Sulfate Streptomycin على B. Subtilis ATCC 21332 التخفيف مقايسة باستخدام

الزيوت. في وقت الحق ..mg/ml2و %3..1 ،(v/v) 0.2% (v/v)، مما أدىالجزئي

C.flexuosus وC. nardus األساسية عندMIC...3 وأضيف كل مرحلة متخلفة من خالل ،

، مما أدى إنتاج البروتينات داخل الخاليا الجديدة مع حجم تقريبي مماثلة C°13نمو البكتيريا عند

كيلو دالتون التي يعترف بها الحمض النووي الموجه الحمض النووي الريبي بوليميراز .38من

الى جانب . انزيم الوحيدات على التوالي αانزيم اختزال النترات والجهاز التنفسي βالوحيدات

من الضروري النفط C. flexuosusقبل تعزيز مع B. Subtilis ATCC 21332 ذلك،

ويمكن أيضا أن .C°1وكبريتات الستربتوميسين خالل مرحلة السجل من الخاليا تنمو بمعدل

كيلو دالتون، كما اعترف .1تفرز البروتينات خارج الخلية مع حجم تقريبي من

Bacillopeptidase F . وباإلضافة إلى ذلك، يمكنB. subtilis ATCC 21332 في وجود

تفرز البروتينات خارج الخلية النشطة الزيوت األساسية C.flexuosus أو C. nardusأو إما

بيولوجيا مع نشاط مضادات الميكروبات قوية ضد البكتيريا إيجابية الجرام وسالبة الجرام

د إما في حالة مرهقة مع وجوB. Subtilis ATCC 21332 وبالتالي، . المحددة

Cymbopogon وكانت الزيوت العطرية أو كبريتات الستربتوميسين عند . ليرة سورية

MIC...3 مستوى قادرة على حمل إنتاج أو إفراز البروتينات النشطة

ix

TABLE OF CONTENT

CONTENTS

Page

AUTHOR DECLARATION i

APPROVAL ii

DEDICATION iii

BIODATA OF AUTHOR iv

ACKNOWLEDMENTS v

ABSTRAK vi

ABSTRACT vii

MULAKHKHAS AL-BAHTH viii

TABLE OF CONTENT ix

LIST OF TABLES xv

LIST OF FIGURES xvi

LIST OF APPENDICES xviii

ABBREVIATIONS xix

CHAPTER I: INTRODUCTION 1

CHAPTER II: LITERATURE REVIEW 4

2.1 Essential Oils 4

2.1.1 Antimicrobial Properties of Essential

Oils

5

2.1.2 The Use of Essential Oils 6

x

2.2 Cymbopogon sp. 7

2.2.1 Cymbopogon flexuosus 7

2.2.1.1 Constituents of Cymbopogon

flexuosus Essential Oil

8

2.2.1.2 Use of Cymbopogon flexuosus

Essential Oil

8

2.2.2 Cymbopogon nardus 9

2.2.2.1 Constituents of Cymbopogon

nardus Essential Oil

9

2.2.2.2 Use of Cymbopogon nardus

Essential Oil

10

2.3 Antibiotics as Antimicrobial Drugs 10

2.3.1 General Characteristics of Antimicrobial

Drugs

10

2.3.2 Streptomycin Sulfate 11

2.4 Stress Responses of Bacteria 13

2.5 Antimicrobial Effects on Bacterial Transcription 14

2.6 Protein 17

2.6.1 Intracellular Protein 18

2.6.2 Extracellular Protein 18

2.7 Peptide 19

2.8 Antimicrobial Protein or Peptide 20

2.9 Bacillus subtilis 21

2.9.1 Protein and Peptide Production 22

2.9.2 Application to Biotechnology 24

CHAPTER III: PROTEIN PRODUCED BY Bacillus subtilis

ATCC 21332 IN THE PRESENCE OF

Cymbopogon nardus ESSENTIAL OIL

28

3.1 Introduction 28

3.2 Materials and Methods 30

3.2.1 Preparation of Essential oil as Stress

Inducer

30

xi

3.2.2 Preparation of Bacterial Strain for

Protein Production

30

3.2.3 Preparation of Test Microorganisms for

Antimicrobial Activity

30

3.2.4 Determination of Inhibitory Effect via

Disc Diffusion Test

31

3.2.5 Determination of Minimum Inhibition

Concentration (MIC)

31

3.2.6 Growth Curve Study 32

3.2.7 Intracellular Proteins Production and

Extraction

32

3.2.8 Extracellular Proteins Production and

Extraction

33

3.2.9 Analysis of Intracellular and

Extracellular Proteins by Sodium

Dodecyl Sulfate-Polyacrylamide Gel

Electrophoresis (SDS-PAGE)

34

3.2.10 Identification of Intracellular Proteins

by Liquid Chromathography-Tandem

Mass Spectrometry (LC-MS/MS)

34

3.2.11 Antimicrobial Activity of Extracellular

Protein

35

3.3 Results 35


Disc Diffusion Test

35


Concentration (MIC)

36


3.3.4 Analysis of Intracellular Proteins by

Sodium Dodecyl Sulfate-

Polyacrylamide Gel Electrophoresis

(SDS-PAGE)

39

3.3.5 Analysis of Extracellular Proteins by



(SDS-PAGE)

42

xii

3.3.6 Identification of Intracellular Proteins



42


Proteins

46

3.4 Discussion 49

3.5 Conclusion 52

CHAPTER IV: PRODUCTION OF PROTEIN BY Bacillus

subtilis ATCC 21332 IN THE PRESENCE

OF Cymbopogon flexuosus ESSENTIAL OIL

54

4.1 Introduction 54


4.2.1 Preparation of Essential oil as Stress

Inducer

56


Protein Production

56

4.2.3 Preparation of Test Microorganisms for

Antimicrobial Activity

56


Disc Diffusion Test

57


Concentration (MIC)

57


4.2.7 Intracellular Proteins Production and

Extraction

58


Extraction

59

4.2.9 Analysis of Intracellular and

Extracellular Proteins by Sodium

Dodecyl Sulfate-Polyacrylamide Gel

Electrophoresis (SDS-PAGE)

60

4.2.10 Identification of Intracellular and

Extracellular Proteins by Liquid

Chromathography-Tandem Mass

Spectrometry (LC-MS/MS)

60


Protein

61

xiii

4.3 Results 62


Disc Diffusion Test

62


Concentration (MIC)

62


4.3.4 Analysis of Intracellular Proteins by



(SDS-PAGE)

65




(SDS-PAGE)

68

4.3.6 Identification of Intracellular and

Extracellular Proteins by Liquid

Chromathography-Tandem Mass

Spectrometry (LC-MS/MS)

72


Proteins

72

4.4 Discussion 75

4.5 Conclusion 81

CHAPTER V: EXTRACELLULAR PROTEIN

PRODUCTION BY Bacillus subtilis ATCC

21332 IN THE PRESENCE OF

STREPTOMYCIN SULFATE

82

5.1 Introduction 82


5.2.1 Preparation of Streptomycin Sulfate

Solution as Stress Inducer

84


Protein Production

84


Disc Diffusion Test

84


Concentration (MIC)

85

xiv



Extraction

86




(SDS-PAGE)

86

5.28 Identification of Extracellular Proteins



87

5.3 Results 87


Disc Diffusion Test

87


Concentration (MIC)

88





(SDS-PAGE)

91

5.3.5 Identification of Extracellular Proteins



95

5.4 Discussion 95

5.5 Conclusion 100

CHAPTER VI: SUMMARY 101

BIBLIOGRAPHY 103

APPENDICES 111

LIST OF PUBLICATIONS 120

xv

xv

LIST OF TABLES

Page

Table 1: Extracellular proteins of B. subtilis 168 26

Table 2: Diameter of inhibition zone (mm) for inhibitory effect of C.

nardus essential oil toward B. subtilis ATCC 21332 by disc

diffusion test

37

Table 3: The Minimum Inhibition Concentration (MIC) of C. nardus

essential oil against B. subtilis ATCC 21332

38

Table 4: Spectrum of antimicrobial activity of extracellular proteins

produced by B. subtilis ATCC21332 prior to enhancing with C.

nardus essential oil

48

Table 5: Diameter of inhibition zone (mm) for inhibitory effect of C.

flexuosus essential oil toward B. subtilis ATCC 21332 by disc

diffusion test

63

Table 6: The Minimum Inhibition Concentration (MIC) of C. flexuosus

essential oil against B. subtilis ATCC 21332

64

Table 7: Spectrum of antimicrobial activity of extracellular proteins

produced by B. subtilis ATCC21332 after inducing with C.

flexuosus essential oil

76

Table 8: Diameter zone of inhibition (mm) for antibacterial test of

Streptomycin Sulfate by disc diffusion method

89

Table 9: Minimum Inhibition Concentration (MIC) of Streptomycin

Sulfate on B. subtilis ATCC 21332

90

xvi

LIST OF FIGURES

Page

Figure 1: The structural formula of Streptomycin Sulfate 12

Figure 2: Concentration dependent effect of antibiotics on bacterial

transcription

16

Figure 3: Protein export pathways in B. subtilis 25

Figure 4: Zone of inhibition produced by Disc Diffusion Test 37

Figure 5: Determination of bacterial viability by using MTT as an

indicator

38

Figure 6: Growth curve of B. subtilis ATCC 21332 for 24 h incubation at

30°C

40


37°C

40

Figure 8: SDS-PAGE profile for intracellular proteins produced by

Bacillus subtilis ATCC 21332 after 24h of incubation

41

Figure 9: SDS-PAGE profile for intracellular proteins produced by

Bacillus subtilis ATCC 21332 after 48 h of incubation

43

Figure 10: SDS-PAGE profile for extracellular protein secreted by Bacillus

subtilis ATCC 21332 after 48 h of incubation

44



45

Figure 12: Peptide sequences of intracellular protein produced by B.

subtilis ATCC 21332 via treatment with C. nardus essential oil

47

Figure 13: The inhibition zone produced by well diffusion test 48



indicator

64


30°C

66

xvii

Page


37°C

66

Figure 18: SDS-PAGE profile for intracellular protein produced by

Bacillus subtilis ATCC 21332 after 24h of incubation

67

Figure 19: SDS-PAGE profile for intracellular protein produced by


69



70



71

Figure 22: Peptide sequences of intracellular protein produced by B.

subtilis ATCC 21332 via treatment with C. flexuosus essential

73

Figure 23: Peptide sequences of extracellular protein produced by B.

subtilis ATCC 21332 via treatment with C. flexuosus essential

oil

74

Figure 24: The inhibition zone produced by well diffusion test 76



indicator

90


30°C

92

Figure 28: SDS-PAGE profile for extracellular protein produced by


93

Figure 29: SDS-PAGE profile for extracellular protein produced by


94

Figure 30: Peptide sequences of extracellular protein produced by B.

subtilis ATCC 21332 via treatment with Streptomycin Sulfate

96

xviii

LIST OF APPENDICES

Page

Appendix A: Serial Dilution of Cymbopogon sp. Essential Oils for Disc

Diffusion Test

111

Appendix B: Serial Two Fold Dilution of Cymbopogon sp. Essential Oils for

MIC Determination

112

Appendix C: McFarland Standard Chart 113

Appendix D: Preparation of C. nardus and C. flexuosus essential oils at 0.01

MIC

114

Appendix E: SDS-PAGE Sample Preparation 115

Appendix F: SDS PAGE Running Buffer 116

Appendix G: The Optical Density (OD) reading of B. subtilis ATCC 21332

culture growth in MHB at 30°C

117

Appendix H: The Optical Density (OD) reading of B. subtilis ATCC 21332

culture growth in MHB at 37°C

118

Appendix I: Preparation of Streptomycin Sulfate Solution : Serial Dilution

119

xix

ABBREVIATIONS

ATCC American Type Culture Collection

ATP Adenosine triphosphate

BaCl2 Barium Chloride

BLAST Basic Local Alignment Search Tool

cfu Colony forming unit

Cm centimeter

dH2O Distilled water

DNA Deoxyribonucleic acid

EO Essential oil

GRAS Generally recognise as safe

h Hour

HCl Hydrogen Chloride

H2SO4 Sulfuric Acid

HPLC High pressure liquid chromatography

kDa kiloDalton

L liter

LC-MS/MS Liquid chromatography-Tandem mass spectrometry

Mg milligram

MHA Mueller-Hinton Agar

MHB Mueller-Hinton Broth

MIC Minimum Inhibitory Concentration

min Minute

ml Milliliter

mm Millimeter

mRNA Messenger ribonucleic acid

MS Mass spectrometer

MTT 3-(4, 5-dimetylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide

NCBI National Center for Biotechnology Information

Nm nanometer

OD Optical Density

PBS Phosphate-buffered saline

Ppm Part per million

RNA Ribonucleic acid

Sdn. Bhd. Sendirian berhad

SDS Sodium Dodecyl Sulfate

SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

sp. Species

Sp Signal peptide

xx

USIM Universiti Sains Islam Malaysia

v Volt

v/v Volume per volume

w/v Weight per volume

% Percentage

ºC Degree Celsius

μl Microliter

CHAPTER VI

SUMMARY

Cymbopogon sp. (which are C. nardus and C. flexuosus) essential oils and

Streptomycin Sulfate at low concentration (which is 0.01 MIC) could induce the

production of proteins by Bacillus subtilis ATCC 21332. C. flexuosus and C. nardus

essential oils, each was added during lag phase of bacterial growth at 37ºC, resulting

the production of new intracellular proteins with similar approximate size of 180 kDa

in which recognized as DNA-directed RNA polymerase β subunit enzyme and

respiratory nitrate reductase α subunit enzyme respectively.

Besides, B. subtilis ATCC 21332 prior to enhancing with C. flexuosus essential

oil and Streptomycin Sulfate during log phase of growing cells at 30ºC could also

secrete the extracellular proteins with approximate size of 30 kDa, recognized as

Bacillopeptidase F. How these antimicrobial compounds modulate the transcription

process in bacteria remains need to be elucidated.

In addition, B. subtilis ATCC 21332 in the presence either C. nardus or C.

flexuosus essential oils could secrete the bioactive extracellular proteins with potent

antimicrobial activity against selected Gram-positive and Gram-negative bacteria.

Therefore, further study can be done to isolate, purify and optimize the production of

bioactive extracellular proteins as well as to determine and evaluate the biological

activities of this protein.

In summary, this study provides some additional information on metabolite

changes that involved in different cellular processes as a response to environmental

stress, and sheds light on the adaptive process. The response of bacteria to

environmental stress is likely complex, involving a combination of different

regulatory circuits. Thus, further study by using biochemical and other approaches

102

will be required in order to determine the changes in enzymatic activity as well as to

identify the specific molecular pathways that involved in mediating the adaptive

responses towards mild stress condition. Besides, of particular interest for future work

will be the detection of transcription modulation induced by other antimicrobials in

which could provide a unique approach to the screening of other proteins synthesized

by microorganisms.

103

BIBLIOGRAPHY

Adhikari, R. P. & R. P. Novick. 2005. “Subinhibitory cerulenin inhibits

staphylococcal exoprotein production by blocking transcription rather than by

blocking secretion”. Microbiology. Vol. 151. pp. 3059-3069.

Al-Ajlani, M. M., S. M. Abid, Z. Ahmad & S. Hasnain. 2007. “Production of surfactin

from Bacillus subtilis MZ-7 grown on pharmamedia commercial medium”. Microbial

Cell Factories. Vo.l 6. pp. 17.

Amanda, S. M., C-O. Florencia & B. Adriano. 2004. “Screening for antimicrobial

activity among bacteria isolated from the Amazon Basin”. Brazilian Journal of

Microbiology. Vol. 35. pp. 307-310.

Andreu, D. & L. Rivas. 1998. “Animal antimicrobial peptides: An overview”.

Biopolymers Peptide Science. Vol. 47. pp. 415-433.

Andrews, J. M. 2001. “Determination of Minimum Inhibitory Concentrations”.

Journal of Antimicrobial Chemotherapy. Vol. 48. pp. 5-16.

Ann, M. S. M., X. Jiru, E. M. John, S. B. Ian & A. M. David. 2007. “Environmental

Stress and Antibiotic Resistance in Food-Related Pathogens”. Applied and

Environmental Microbiology. Vol. 73. pp. 211-217.

Asthana, A., R. A. Larson, K. A. Marley, R. W. Tuveson. 1992. “Mechanism of citral

phototoxicity”. Phytotoxicity and Photobiology. Vol. 56. pp. 211-222.

Bal, S., R. R, Mishra, B. Rath, H. K. Sahu & H. N. Thatoi. 2009. “Characterization

and extracellular enzyme activity of predominant marine Bacillus spp. isolated from

sea water of Orissa Coast, India”. Malaysian Journal of Microbiology. Vol. 5. pp. 87-

93.

Başer, K.K.C & F. Demirci. 2007. “Chemistry of essential oils”. Flavours and

Fragrances. Vol. 5. pp. 43-86.

Bauer, K., D. Garbe, H. Surburg. 2001. Common Fragrance and Flavor Materials:

Preparation, Properties and Uses – 4. ed. Weinheim: Wiley-VCH.

Beisswenger, C. & R. Bals. 2005. “Functions of antimicrobial peptides in host defense

and immunity”. Current Protein and Peptide Science. Vol. 6. pp. 255-264.

Bertea, C. M. & M. E. Maffei. 2010. “The Genus Cymbopogon: Botany, including

anatomy, physiology, biochemistry, and molecular biology”. Essential Oil Bearing

Plants: The genus Cymbopogon. Edited by: Anand Akhila. Boca Raton, FL: CRC

Press Taylor & Francis Group.

Black, J. G., 2005. Microbiology: Principles and Explorations – 6. ed. USA: John

Wiley & Sons.

104

Blazquez, J., J. M. Gomez-Gomez, A. Oliver, C. Juan, V. Kapur, S. Martin. 2006.

“PBP3 inhibition elicits adaptive responses in Pseudomonas aeruginosa”. Molecular

Microbiology. Vol. 62. pp. 84-99.

Boor, K. J., M. L. Duncan & C. W. Price. 1995. “Genetic and transcriptional

organization of the region encoding the β subunit of Bacillus subtilis RNA

polymerase”. Journal of Biological Chemistry. Vol. 270. pp. 20329-20336.

Boor, K. J. 2006. “Bacterial Stress Responses: What Doesn’t Kill Them Can Make

Them Stronger”. PLoS Biology. Vol. 4. pp. 18-20.

Borman. 2007. “Protein Factory Reveals Its Secrets”. Chemistry. Vol. 85. pp. 13-16.

Brenda, Y. R., R. C. Franklin, B. P. Daniel, W. S. Daniel & L. L. Mark. 2004.

“Effects of sub-minimum inhibitory concentration antibiotic levels and temperature on

growth kinetics and outer membrane protein expression in Mannheimia haemolytica

and Haemophilus somnus”. The Canadian Journal of Veterinary Research. Vol. 69.

pp. 1-10.

Burt, S.A. R. van deer Zee, A. P. Koets, A. M. de Graaff, F. van Knapen, W. Gaastra,

H. P. Haagsman & J. A. Veldhuizen. 2007. “Carvacrol induces heat shock protein 60

and inhibits synthesis of flagellin in Escherichia coli 0157:H7”. Applied and

Environmental Microbiology. Vol. 73. pp. 4484-4490.

Burt, S. 2004. “Essential oils: their antibacterial properties and potential applications

in foods—a review”. International Journal of Food Microbiology. Vol. 94. pp. 223-

253.

Carter, G., L. S. Young & L. E. Bermudez. 2004. “A subinhibitory concentration of

clarithromycin inhibits Mycobacterium avium biofilm formation”. Antimicrobial

Agents and Chemotheraphy. Vol. 48. pp. 4907-4910.

Chang, S. C. & L. J. Cseke. 2004. “Extraction and purification of proteins”. Handbook

of Molecular and Cellular in Biology and Medicine. Vol. 3. pp 48-49.

Cosentino, S., C. I. G. Tuberoso, B. Pisano, M. Satta, V. Mascia, E. Arzedi & F.

Palmas. 1999. “In-vitro antimicrobial activity and chemical composition of Sardinian

thymus essential oils”. Letters in Applied Microbiology. Vol. 29. pp. 130-135.

Cowen, L. E. & W. J. Steinbach. 2008. “Stress, Drugs, and Evolution: the Role of

Cellular Signaling in Fungal Drug Resistance”. American Society for Microbiology.

Vol. 7. pp. 47-764.

Davies, J., G. B. Spiegelman & G. Yim. 2006. “The world of subinhibitory antibiotic

concentrations”. Current Opinion in Microbiology. Vol. 9. pp. 445-453.

105

Eloff, J.N. 1998. “A sensitive and quick microplate method to determine the minimal

inhibitory concentration of plants extract for bacteria”. Plant Medica. Vol. 6. pp. 711-

713.

Fajardo, A. & Martı´nez. 2008. “Antibiotics as signals that trigger specific bacterial

responses”. Current Opinion in Microbiology. Vol. 11. pp. 161-167.

Fernandez de Caleya, R., B. Gonzalez-Pascual, F. García-Olmedo & B. Carbonero.

1972. “Susceptibility of phytopathogenic bacteria to wheat purothionins in vitro”.

Applied Microbiology. Vol. 23. pp. 998-1000.

Finken, M., P. Kirschner, A. Meier, A. Wrede & E. C. Böttger. 1993. “Molecular

basis of streptomycin resistance in Mycobacterium tuberculosis: Alterations of the

ribosomal protein S12 gene and point mutations within a functional 16S ribosomal

RNA pseudoknot”. Molecular Microbiology. Vol. 9. pp. 1239-1246.

Finlay, B. B. & R. E. W. Hancock. 2004. “Can innate immunity be enhanced to treat

microbial infections?”. Nature Reviews Microbiology. Vol. 2. pp. 497–504.

Friedrich, W. 2010. “Theory and Measurement of Bacterial Growth”. Online

Microbiology Notes. www.mpi-bremen.de/Binaries/Binary13037/Wachtstumversuch.

Gerald, K. 2001. Cell and Molecular Biology : Concepts and Experiments – 10. ed.

USA: John Wiley & Sons.

Gibson, G. & Muse, S.V. 2009. A Primer of Genome Science – 3. ed. USA : Sinauer

Associates .

Goh, E-B., G. Yim, W. Tsui, J. McClure, M. G. Surette & J. Davies. 2002.

“Transcriptional modulation of bacterial gene expression by subinhibitory

concentrations of antibiotics”. Proceeding of National Academic Science. Vol. 99. pp.

17025-17030.

Gould, G. W. 1989. “Heat-induced injury and inactivation”. Applied Science. Vol. 2.

pp. 11- 42.

Hancock, R. E. W. & R. Lehrer . 1998. “Cationic peptides: a new source of

antibiotics”. Trends in Biotechnology. Vol. 16. pp. 82-88.

Hanina, M. N., M. N. Nurul-Aini, I. Nazlina, M. D. Yahya, A. R. Saziah, I. M. Said &

I. B. Ahmad. 2002. “Antimicrobial activity of Cymbopogan nardus (L.) Rendle

extract fractions against Staphylococcus aureus”. Malaysian Applied Biology. Vol. 31.

pp. 63-65.

Harwood, C. R. 1992. “Bacillus subtilis and its relatives: molecular biological and

industrial workhorses”. Trends Biotechnology. Vol. 10. pp. 247-256.

http://www.mpi-bremen.de/Binaries/Binary13037/Wachtstumversuch

106

Henderson-Begg, S. K., D. M. Livermore & L. M. Hall. 2006. “Effect of subinhibitory

concentrations of antibiotics on mutation frequency in Streptococcus pneumoniae.

Journal of Antimicrobials and Chemotheraphy. Vol. 57. pp. 849-854.

Hoffman, L. R., D. A. D’Argenio, M. J. MacCoss, Z. Zhang, R. A. Jones & S. I.

Miller. 2005. “Aminoglycoside antibiotics induce bacterial biofilm formation”.

Nature. Vol. 436. pp.1171-1175.

Jaganath, I.B. & L. T. Ng. 2000. Herbs: The Green Pharmacy of Malaysia – 1. ed.

Malaysia: Vinpress Sdn. Bhd.

Kalemba, D. & A. Kunicka. 2003. “Antibacterial and antifungal properties of essential

oils”. Current Medicinal Chemistry. Vol. 10. pp. 813-829.

Kevin, M. & Devine. 2004. The Desk Encyclopedia Of Microbiology – 1 ed. United

Kingdom: Academic Press.

Kim, Y., M. Coppey, R. Grossman, L. Ajuria, G. Jimmenez, S. Paroush &

Shvartsman. 2010. “Antibacterial activity of some essential oil components against

five foodborne pathogens”. Current Biology. Vol. 20. pp. 1-6.

Korsten, L. & N. Cook. 1996. “Optimizing Culturing Conditions for Bacillus

Subtilis”. South African Avocado Growers Association Yearbook. Vol. 19. pp. 54-58.

Kwon, G-H., P. Jae-Yong, K. Jong-Sang, L. Jinkyu, P. Cheon-Seok, Y. K. Dae & H.

K. Jeong. 2011. “Cloning and Expression of a bpr Gene Encoding Bacillopeptidase F

from Bacillus amyloliquefaciens CH86-1”. Journal of Microbiology & Biotechnology.

Vol. 21. pp. 515-518.

Laemmli, U. K. 1970. “Cleavage of Structural Proteins during the Assembly of the

Head of Bacteriophage T4”. Nature. Vol. 227. pp. 680-685.

Lash, B. W., T. H. Mysliwiec & H. Gourama. 2005. “Detection and partial

characterization of a broad-range bacteriocin produced by Lactobacillus plantarum

(ATCC 8014)”. Food Microbiology. Vol. 22. pp. 199-204.

Lau, S.KP., R. YY. Fan, T. CC. Ho, G. KM. Wong, A. KL. Tsang, J. LL. Teng, W.

Chen, R. M. Watt, S. OT. Curreem, H. Tse, K. Y. Yuen & P. CY. Woo. 2011.

“Environmental adaptability and stress tolerance of Laribacter hongkongensis: a

genome-wide analysis”. Cell & Bioscience. Vol. 1. pp. 22.

Leclere, V. M. Bechet, A. Adam, J-S. Guez, B. Wathelet, M. Ongena, P. Thonart, F.

Gancel, M. Chollet-Imbert & P. Jacques. 2005. “Mycosubtilin overproduction by

Bacillus subtilis BBG100 enhances the organism’s antagonistic and biocontrol

activities”. Applied and Environmental Microbiology. Vol. 71. pp. 4577-4584.

Mackintosh, J. A., D. A. Veal, A. J. Beattie, & A. A. Gooley. 1998. “Isolation from an

ant Myrmecia gulosa of two inducible O-glycosylated proline-rich antibacterial

peptides”. Journal of Biological Chemistry. Vol. 273. pp. 6139-6143.

107

Madigan, M. T. & J. M. Martinko. 2006. Brock Biology of Microorganisms – 8. ed.

USA : Pearson Prentice Hall.

Marino, M. 2001. “Modulation of anaerobic energy metabolism of Bacillus subtilis by

arfM (ywiD)”. Journal of Bacteriology. Vol. 183. pp. 6815-6821.

Mitova, M. I., G. Lang, J. Wiese & J. F. Imhoff. 2008. “Subinhibitory concentrations

of antibiotics induce phanazine production in a marine Streptomyces sp.”. Journal of

Natural Product. Vol. 71. pp. 824-827.

Moore, S. J., & A. D. Lenglet. 2004. “An overview of plants used as insect

repellents”. In: Insect Repellents: Principles, Methods, and Use. (Debboun, M., S. P.

Frances & D. Strickman eds.) CRC Press, Taylor & Francis Group Boca Raton

Florida. pp. 343-363.

Moszer, I., P. Glaser & A. Danchin. 1995. “A relational database for the Bacillus

subtilis genome”. Microbiology. Vol. 141. pp. 261-268.

Murray, M. Y. 2011. Comprehensive Biotechnology–III : Protein 2. ed. Elsevier B.V.

Nakahara, K., N. J. Alzoreky, T. Yoshihashi, H. T. T. Nguyen & G. Trakoontivakorn.

2003. “Chemical Composition and Antifungal Activity of Essential Oil from

Cymbopogon nardus (Citronella Grass)”. Japan Agricultural Research Quarterly.

Vol. 37. pp. 249-252.

Nakano, M. M. & P. Zuber. 1998. “Anaerobic Growth of a “Strict Aerobe” (Bacillus

Subtilis)”. Annual Review of Microbiology. Vol. 52. pp. 165-190.

Omura, K., M. Hitosugi, X. Zhu, M. Ikeda, H. Maeda & S. Tokudome. 2005. “A

Newly Derived Protein from Bacillus subtilis natto with Both Antithrombotic and

Fibrinolytic Effects”. Journal of Pharmacological Sciences. Vol. 99. pp. 247-251.

Oosterhaven K, B. Poolman, E. J. Smid. 1995. “S-Carvone as a natural potato sprout

inhibiting, fungistatic and bacteriostatic compound”. Industrial Crops and Products.

Vol. 4. pp. 23-31.

Oussalah, M., S. Caillet, L. Saucier & M. Lacroix. 2006. “Antimicrobial effects of

selected plant essential oils on the growth of a Pseudomonas putida strain isolated

from meat”. Meat Science. Vol. 73. pp. 236-244.

Oyen, L. P. A & X. D. Nguyen. 1999. Plant Resources of South-East Asia No.19:

Essential Oil Plants. Netherlands : Backhuys Publishers.

Ozkalp B. & M M. Ozcan. 2009. “Inhibitory effect of hydrodistillation waters of some

medicinal and aromatic plants”. World Applied Science Journal. Vol. 6. pp. 825-828.

108

Papo, N. & Y. Shai. 2003. “Can we predict biological activity of antimicrobial

peptides from their interactions with model phospholipid membranes?”. Peptides. Vol.

24. pp. 1693-1703.

Piggot, P. J. 2009. Encyclopedia of Microbiology – 3. ed. Elsevier Inc.

Powers, J-P. S. & R. E. W. Hancock. 2003. “The relationship between peptide

structure and antibacterial activity”. Peptides. Vol. 24. pp. 1681-1691.

Prabuseenivasan, S., M. Jayakumar & S. Ignacimuthu. 2006. “In vitro antibacterial

activity of some plant essential oils”. BMC Complementary and Alternative Medicine.

Vol. 6. pp. 39.

Rifkind, D. & G. L. Freeman. 2005. The Nobel Prize Winning Discoveries in

Infectious Diseases. pp 47-50.

Rizal, M.A. 2008. “Antimicrobial effect of essential oil from Cymbopogon flexuosus”.

Bachelor of Science with Honours thesis, Universiti Sains Islam Malaysia.

Rowbury, R. J. 1998. “Life science update: Do we need to rethink our ideas on the

mechanisms of inducible processes in bacteria?”. Science Programme. Vol. 81. pp.

193.

Scott, T. & M. Eagleson, M. 1988. Concise Encyclopedia: Biochemistry - 2 ed. USA:

Walter de Gruyter.

Shinde, V. A., S. M. More & T. A. Kadam. 2012. “Antimicrobial Activity Of

Phospholipid Compound Produced by Alkaliphilic Bacillus subtilis Isolated from

Lonar Lake”. International Journal of Innovations in Bio-Sciences. Vol. 2. pp. 172-

175.

Sikder, M. A. & S. Kenji. 2009. “Lantibiotics: Diverse activities and unique modes of

action”. Journal of Bioscience and Bioengineering. Vol. 107. pp. 475-487.

Singh, A., R. K. Singh, A. K. Bhunia & N. Singh. 2003. “Efficacy of plant essential

oils as antimicrobial agents against Listeria monocytogenes in hotdogs”. Lebensm.-

Wiss. u.-Technology. Vol. 36. pp. 787-794.

Singh, S., M. Ram, D. Ram, V. P. Singh, S. Sharma & Tajuddin. 2000. “Response of

lemongrass (Cymbopogon flexuosus) under different level of irrigation on deep sandy

soils”. Irrigation Science. Vol. 20. pp. 15-21.

Sims, G. K. 2006. “Nitrogen starvation promotes biodegradation of N-heterocyclic

compounds in soil”. Soil Biology and Biochemistry. Vol. 38. pp. 2478-2480.

Smith-Palmer, A., J. Stewart & L. Fyfe. 1998. “Antimicrobial properties of plant

essential oils and essences against five important food borne pathogens”. Letters in

Food Microbiology. Vol. 26. pp. 118-122.

109

Strahl, E. D., W. E. Dobson & L. L. Lundie. 2002. “Isolation and screening of brittle

star associated bacteria of antibacterial activity”. Current Microbiology. Vol. 44. pp.

450-459.

Tanaka, M., T. Hasegawa, A. Okamoto, K. Torii & M. Ohta. 2005. “Effect of

Antibiotics on Group A Streptococcus Exoprotein Production Analyzed by Two-

Dimensional Gel Electrophoresis”. Antimicrobial Agents and Chemotherapy. Vol. 49.

pp. 88-96.

Tjalsma, H., H. Antelmann, J. D. H. Jongbloed, P. G. Braun, E. Darmon, R. Dorenbos,

J. Y. F. Dubois, H. Westers, G. Zanen, W. J. Quax, O. P. Kuipers, S. Bron, M. Hecker

& J. M. van Dijl. 2004. “Proteomics of Protein Secretion by Bacillus subtilis:

Separating the “Secrets” of the Secretome”. Microbiology and Molecular Biology

Review. Vol. 68. pp. 207-233.

Tsao, R. & T. Zhou. 2007. “Natural antimicrobials from plant essential oils”. ACS

Symposium Series. Vol. 967. pp. 364-387.

Tzortzakis, N. G. & C. D. Economakis. 2007. “Antifungal activity of lemongrass

(Cympopogon citratus L.) essential oil against key postharvest pathogens”. Innovative

Food Science & Emerging Technologies. Vol. 8. pp. 253-258.

Vaara, M. 1992. “Agents that increase the permeability of the outer membrane”.

Microbiology Review. Vol. 56. pp. 395.

Van de Braak, S. A. A. J. & G. C. J. J. Leijten. 1999. “Essential Oils and Oleoresins:

A Survey in the Netherlands and other Major Markets in the European Union”. CBI,

Centre for the Promotion of Imports from Developing Countries, Rotterdam. pp. 116.

Vardanyan, R. S. & V. J. Hruby. 2006. Synthesis of Essential Drugs. pp 425-498.

Vijayalakshmi, K. & S. Rajakumar. 2010. “Antimicrobial protein production by

Bacillus amyloliquefaciens MBL27: An application of statistical optimization

technique”. African Journal of Microbiology Research. Vol. 4. pp. 2388-2396.

Wander, M. 2002. “Proteolytic activity under nitrogen or sulfur limitation”.

Application of Soil Ecology. Vol. 568. pp. 1-5.

Wong, S. l. 1995. “Advances in the use of Bacillus subtilis for the expression and

secretion of heterologous proteins”. Current Opinion in Biotechnology. Vol. 6. pp.

517-522.

Wright, J. M. & R. J. Lewis. 2007. “Stress Responses of Bacteria”. Current Opinion

in Structural Biology. Vol. 17. pp. 755-760.

Wu, X. C., W. Lee, L. Tran & S. L Wong. 1991. “Engineering a Bacillus subtilis

expression-secretion system with a strain deficient in six extracellular proteases”.

Journal of Bacteriology. Vol. 173. pp. 4952-4958.

110

Yamagata, Y., R. Abe, Y. Fujita & E. Ichishima. 1995. “Molecular cloning and

nucleotide sequence of the 90k serine protease gene, hspK, from Bacillus subtilis

(natto) No. 16”. Current Microbiology. Vol. 31. pp. 340-344.

Yang, X. & C. W. Price. 1995. “Streptolydigin resistance can be conferred by

alterations to either the β and β’ subunits of Bacillus subtilis RNA polymerase”.

Journal of Biological Chemistry. Vol. 270. pp. 23930-23933.

Yim, G., H. H. Wang & FRS. J. Davies. 2007. “Antibiotics as signalling molecules”.

Philosophical Transactions of the Royal Society Biological Sciences. Vol. 362. pp.

1195-1200.

Yoshinori, M. & S. Fereidoon. 2006. Nutraceutical Proteins and Peptides in Health

and Disease – 1 ed. London : Taylor & Francis Group.

Zakiah, M. 2008. “Antimicrobial activity of essential oils from Cymbopogon nardus”.

Bachelor of Science with Honours thesis. Universiti Sains Islam Malaysia.

Zierhut, G., W. Piepersberg, A. Böck. 1979. “Comparative analysis of the effect of

aminoglycosides on bacterial protein synthesis in vitro”. European Journal of

Biochemistry. Vol. 98. pp. 577-583.

111

APPENDIX A

Serial Dilution of Cymbopogon sp. Essential Oils for Disc Diffusion Test

Concentration % (v/v)

Volume of Essential Oils

(μl)

Volume of MHB

(μl)

1:0 (100) 1000 -

1:1 *(50.0) a100

b100

1:2 (33.3) 100 200

1:3 (25.0) 100 300

1:4 (20.0) 100 400

1:5 (16.7) 100 500

1:6 (14.3) 100 600

1:7 (12.5) 100 700

1:8 (11.1) 100 800

1:9 (10.0) 100 900

*Calculation for concentration

aVolume of essential oils x 100% = %

aVolume of essential oils +

bVolume of MHB

e.g. : 100 x 100% = 50%

100 + 100

112

APPENDIX B

Serial Two Fold Dilution of Cymbopogon sp. Essential Oils for MIC Determination

100% Essential Oil (EO)

25% 50% 12.5% 6.25%

0.39% 0.78% 1.56% 3.13%

0.2% 0.098% 0.049% 0.024%

500μl of EO + 500 μl 10% DMSO

500μl of 12.5% EO +

500μl 10% DMSO

500μl of 25% EO + 500μl 10% DMSO

500μl of 50% EO + 500μl 10% DMSO

500μl of 0.2% EO +

500μl 10% DMSO

500μl of 3.13% EO + 500μl 10% DMSO

500μl of 1.56% EO +

500μl 10% DMSO

500μl of 6.25% EO + 500μl 10% DMSO

500μl of 0.39% EO +

500μl 10% DMSO

500μl of 0.78% EO +

500μl 10% DMSO

500μl of 0.098% EO

+ 500μl 10% DMSO

500μl of 0.049% EO

+ 500μl 10% DMSO

113

APPENDIX C

McFarland Standard Chart

BaCl2 H2SO4 Approx. cfu/ml

(x108)

Optical Density

0.1 9.9 3 0.180

0.2 9.8 6 0.310

0.3 9.7 9 0.485

0.4 9.6 12 0.605

0.5 9.5 15 0.708

0.6 9.4 18 0.883

0.7 9.3 21 1.030

0.8 9.2 24 1.212

0.9 9.1 27 1.225

1.0 9.0 30 1.325

114

APPENDIX D

Preparation of C. nardus and C. flexuosus essential oils at 0.01 MIC

1. Preparation of C. nardus essential oil at concentration of 1 x MIC.

MIC value of C. nardus = 1.56%

1.56 x 2 ml = 0.03 ml

100

Mix 0.03 ml of C. nardus essential oil with 1.97 ml of 10% DMSO solution in

order to prepare 2 ml of 1.56% C. nardus essential oil.

2. Preparation of C. flexuosus essential oil at concentration of 1 x MIC.

MIC value of C. flexuosus = 0.2%

0.2 x 2 ml = 0.004 ml

100

Mix 0.004 ml of C. flexuosus essential oil with 1.996 ml of 10% DMSO

solution in order to prepare 2 ml of 0.2% C. flexuosus essential oil.

3. Preparation of Cymbopogon sp. essential oils at 0.01 x MIC as stress inducer

for B. subtilis ATCC 21332.

1 x MIC

100

Add 0.5 ml of each 1 x MIC Cymbopogon sp. essential oils to 49.5 ml of

bacterial culture in order to get the final concentration of Cymbopogon sp.

essential oils at 0.01 MIC.

115

Appendix E

SDS-PAGE Sample Preparation

Instructions are provided for electrophoresis of Mini-PROTEAN® TGX

TM precast gels

using Mini-PROTEAN Tetra cell system

Reagent Reduced Sample

Sample (pellet) 5 μl

Laemmli Sample Buffer 4.75 μl

β-mercaptoethanol 0.25 μl

Total Volume 10 μl

Add Reducing Agent

Add 0.25 μl of β-mercaptoethanol per 4.75 μl of Bio-Rad’s Laemmli sample buffer for

a final concentration of 5% β-mercaptoethanol, 710 mM.

Note: For best results, do not store Bio-Rad’s Laemmli sample buffer with β-

mercaptoethanol.

Dilute Sample

Dilute 1 part sample with 1 part Laemmli sample buffer (1:1).

Heat samples at 90-100°C for 5 min

116

APPENDIX F

SDS PAGE Running Buffer

Components Quantity

Tris – base 6.04 g

Glycine 28.8 g

SDS 2 g

Distilled H20 1.8 L

1. Dissolve Tris base and glycine together in 1.8 L of ddH2O.

2. Add SDS and mix.

3. Add dH2O to a final volume of 2 L.

117

APPENDIX G

The Optical Density (OD) reading of B. subtilis ATCC 21332 culture growth in MHB

at 30°C

Time (h) Optical Density (OD)

0 0.03

1 0.03

2 0.03

4 0.04

6 0.07

8 0.13

10 0.17

12 0.21

14 0.21

16 0.21

18 0.21

20 0.19

22 0.15

24 0.15

118

APPENDIX H

The Optical Density (OD) reading of B. subtilis ATCC 21332 culture growth in MHB

at 37°C

Time (h) OD

0 0.01

1 0.01

2 0.01

4 0.03

6 0.05

8 0.09

10 0.14

12 0.18

14 0.20

16 0.20

18 0.20

20 0.19

22 0.16

24 0.16

119

APPENDIX I

Preparation of Streptomycin Sulfate Solution : Serial Dilution

aStreptomycin

Sulfate Solutions

(mg/ml)

bFirst dilution

(mg/ml)

cSecond dilution

(mg/ml)

dThird dilution

(mg/ml)

10.0

1.0

0.1

0.01

5.0

0.5 0.05 0.005

2.5 0.25 0.025 0.0025

aStreptomycin Sulfate solutions were prepared accordingly:

(1) 10 mg/ml – 10 mg of Streptomycin Sulfate was added to 1 ml of sterilized

distilled water;

(2) 5 mg/ml – 500 µl of 10 mg/ml antibiotic solution was added to 500 µl of

sterilized distilled water;

(3) 2.5 mg/ml – 500 µl of 5 mg/ml antibiotic solution was added to 500 µl of

sterilized distilled water.

bThe first serial dilution was done by adding 100 µl Streptomycin Sulfate solution (10,

5 or 2.5 mg/ml) into 900 µl sterilized distilled water to give a subsequent final

concentration of 1.0, 0.5 or 0.25 mg/ml.

cThe second serial dilution was done by adding 100 µl Streptomycin Sulfate solution

(1.0, 0.5 or 0.25 mg/ml) into 900 µl sterilized distilled water to give a subsequent final

concentration of 0.1, 0.05 or 0.025 mg/ml.

dThe third serial dilution was done by adding 100 µl Streptomycin Sulfate solution

(0.1, 0.05 or 0.025 mg/ml) into 900 µl sterilized distilled water to give a subsequent

final concentration of 0.01, 0.005 or 0.0025 mg/ml.

120

LIST OF PUBLICATIONS

1) Hanina Mohd Noor, Hairul Shahril Muhamad, Mohd Fazrullah Innsan

Mohd Fauzi, Ismatul Nurul Asyikin Ismail, Abdul Jalil Abdul Kadir,

Salina Mat Radzi & Ismail Bin Ahmad. 2011. “Protein production by Bacillus

subtilis ATCC 21332 in the presence of Cymbopogon essential oils”. World

Academy of Science, Engineering and Technology. Vol. 59. pp. 273-277.

2) Hanina Mohd Noor, Hairul Shahril Muhamad, Ismatul Nurul Asyikin Ismail,

Salina Mat Radzi, Maryam Mohamed Rehan, Abdul Jalil Abdul Kader &

Rosfarizan Mohamad. 2014. “Protein Produced by Bacillus subtilis

ATCC21332 in the Presence of Cymbopogon flexuosus Essential oil”. Key

Engineering Materials. Vols. 594-595. pp. 370-377

3) Hairul Shahril Muhamad, Hanina Mohd Noor, Ismatul Nurul Asyikin Ismail,

Salina Mat Radzi, Abdul Jalil Abdul Kader, Maryam Mohamed Rehan &

Rosfarizan Mohamad. “Protein produced by Bacillus subtilis ATCC 21332 in

the presence of Cymbopogon nardus essential oil”. Proceeding for

International symposium on functional genomics and structural biology 2014.

Universiti Putra Malaysia. pp. 20-21.

4) Hairul Shahril M., Mohd Fazrullah Innsan M. F., Ismatul Nurul Asyikin I.,

Hanina M. N., Abdul Jalil A. K & Salina M. R. “Production Of Protein In The

Presence Cymbopogon flexuosus Essential Oil and Streptomycin Sulphate at

Low Sub Lethal Concentration”. Extended Abstracts of Fundamental Science

Congress 2012. Universiti Putra Malaysia. pp. 18-19.

5) Hairul Shahril M., M.Z. Mohd Shazwan, M.F. Mohd Fazrullah Innsan, S.

Muhammad Nawawi Munir, I. Ismatul Nurul Asyikin, M.R. Salina & M.N

Hanina. “Anti-quorum sensing activity of local ulam in Malaysia”. Proceeding

for International conference on natural product 2011. Universiti Putra

Malaysia. Pp. 15-16.

Date post:	29-Jan-2017
Category:	Documents
Upload:	ngohanh
View:	222 times
Download:	6 times

effects of cymbopogon sp. essential oils and streptomycin sulfate on ...

Documents